The present study examined the fatty acid content of human milk from Polish women living in the Warmia and Mazury region with regard to different lactation periods and compared it with the fatty acid content of selected infant formulas. The analysis included samples of breast milk-colostrum (n = 21), transitional milk (n = 26), and mature milk (n = 22). Fat was extracted using the Rose-Gottlieb method, and the fatty acid profile was determined by gas chromatography with a flame ionization detector (FID). The proportion of SFAs (saturated fatty acids) > MUFAs (monounsaturated fatty acids) > PUFAs (polyunsaturated fatty acids) was determined in each fraction of breast milk and infant formula. Palmitic, oleic, and linoleic acids predominated in breast milk and infant formulas. Colostrum contained lower contents of selected SFAs (caprylic, capric, lauric) and higher contents of selected MUFAs (ercucic) and PUFAs (arachidonic and docosahexaenoic) (p < 0.05) relative to transitional and mature milk. Infant formulas were distinguished from human milk in terms of their SFA (caproic, caprylic, lauric, arachidic), MUFA (oleic), and PUFA (linoleic, α-linoleic) content. It should be noted that infant formulas contained significantly lower trans fatty acid (TFA) content-more than thirty-six and more than nineteen times lower than in human milk. Furthermore, human milk contained branched-chain fatty acids (BCFAs) at 0.23-0.28%, while infant formulas contained only trace amounts of these acids. The average ratio of n-6 to n-3 fatty acids for human milk was 6.59:1 and was close to the worldwide ratio of 6.53 ± 1.72:1. Both principal component analysis (PCA) and cluster analysis (CA) indicated significant differences in the fatty acid profile relative to lactation and a different profile of infant formulas relative to breast milk.